The determination of analytes in samples plays an important role in either environmental or human diagnostics analysis. The infection or pollution of samples by substances coming from the environment is becoming a major focus of the industry. Because many of the substances are present in samples in very low amounts, the methods for the analysis must be very sensitive. This is especially true for immunological determinations or analyses based on the occurrence of nucleic acids.
The first increase in sensitivity to be achieved in nucleic acid assays was realised by the possibility to amplify the amount of analyte nucleic acid in a sample. This has made possible the determination of even very low amounts of nucleic acids present in a sample. One example of a method for the amplification of analyte nucleic acids in a sample is the so-called polymerase chain reaction which is described in detail in U.S. Pat. No. 4,683,202. This method uses two primers chosen such that the sequence of the first primer is complementary to a region of the target nucleic acid to be amplified and the sequence of the second primer is homologous to a sequence on the target nucleic acid such that the elongation product of one primer can be used as a template for the elongation of the other primer. This method yields multiple copies of the nucleic acid to be determined.
As a further development of this method in EP-A 0 379 369 there is described a method for converting an analyte polynucleotide into a polynucleotide having at one terminus a nucleobase sequence which is complementary to the sequence at the other terminus. This newly constructed polynucleotide containing a fragment of the analyte nucleic acid is capable of being amplified using only one primer sequence. This procedure, however, has the disadvantage that the termini of the nucleic acid produced can hybridise to each other and therefore can prevent annealing of the primer. Therefore the amplification is not very effective. Further, the preparation of the amplifyable intermediate product requires the use of two different primer sequences and hence knowledge of two different sequences in the nucleic acid to be determined or amplified.
Therefore, it is the object of the present invention to provide the art with a method for generating multiple double stranded nucleic acids which can be used to determine analytes, where only one primer sequence is used.